The structure of the bacterial nucleoid and the forces which maintain its DNA in a highly compact yet accessible form are largely unknown. We have been systematically characterizing spermidine nucleoids isolated from E. coli under relatively non-denaturing conditions as an approach to these problems. The isolated nucleoids have multiple restraints to DNA unfolding; each restraint is characterized by a urea concentration giving 50% unfolding (Um value) and by the effects of various agents, particularly exogenous RNase. An RNase-sensitive restraint to DNA unfolding with Um=3.2 M urea and an RNase-resistant restraint with Um=1.8 M urea have been partially characterized. The RNase-sensitive restraint appears to be actual linkages between nucleoidal DNA and residual cell envelope which result from cotranslational insertion that occurred in the cells from which the nucleoids were isolated. Such linkages would have the following connections: DNA - RNA polymerase - mRNA - ribosomes - nascent polypeptide - residual cell membrane. Support for such a basis for the RNase-sensitive unfolding at 3.2 M urea comes from 1) effects on the stability of isolated nucleoids resulting from preexposures of cells to antibiotics; antibiotics which specifically inhibit protein or RNA syntheses caused large changes in Um values that were consistent with the proposed linkages; 2) destruction of one component of the linkage, the ribosomes, occurred at ca. 3 M urea in both isolated nucleoids and purified ribosome preparations; the ribosomes may be the specific target removed at this urea concentration; 3) the high sensitivity of nucleoidal mRNA to RNase; 4) retention of the RNase-sensitive unfolding at 3 M urea, despite removal of most DNA-binding proteins (except RNA polymerase) by salt extractions, indicating that the conventional model for DNA compaction by protein binding is not controlling the unfolding at 3 M urea. The other restraint studied, the RNase-resistant restraint, was destroyed by extended treatment of isolated nucleoids with muramidases, indicating a relation to residual murein. - antibiotics,DNA,Escherichia coli,nucleoids,proteins,RNA,urea